This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have synthesized an irreversible inhibitor that binds to a variety of protein kinases. This compound, FSPP, covalently modifies a conserved catalytic lysine active site and carries a propargyl group to attach diverse fluorescent and affinity tags after protein modification via copper catalyzed "click chemistry." The covalent nature of FSPP's inhibitory mechanism facilitates identification of its kinase targets. We are using this compound's chemical properties to probe the kinases of the eukaryotic parasite Trypanosoma brucei, the causative agent of African sleeping sickness, for new potential therapeutic targets. FSPP inhibits T brucei growth at one micro molar, and we are attempting to identify the essential kinase(s) mediating this toxicity using affinity purification and mass spectrometry. Additionally we are using a competitive labeling strategy to identify potential reversible active site directed inhibitors of the same kinases. Non-covalent inhibitors sharing targets with FSPP will slow or halt the rate of those targets'chemical modification when cell lysates are co-treated with FSPP and the reversible competitor. We believe this strategy will identify novel therapeutic targets for this disease while at the same time providing valuable structure activity relationships for future drug development.